Effect of a Novel Trivalent Vaccine Formulation against Acute Lung Injury Caused by Pseudomonas aeruginosa

An effective vaccine against Pseudomonas aeruginosa would benefit people susceptible to severe infection. Vaccination targeting V antigen (PcrV) of the P. aeruginosa type III secretion system is a potential prophylactic strategy for reducing P. aeruginosa-induced acute lung injury and acute mortality. We created a recombinant protein (designated POmT) comprising three antigens: full-length PcrV (PcrV#1-#294), the outer membrane domain (#190-342) of OprF (OprF#190-#342), and a non-catalytic mutant of the carboxyl domain (#406-613) of exotoxin A (mToxA#406-#613(E553Δ)). In the combination of PcrV and OprF, mToxA, the efficacy of POmT was compared with that of single-antigen vaccines, two-antigen mixed vaccines, and a three-antigen mixed vaccine in a murine model of P. aeruginosa pneumonia. As a result, the 24 h-survival rates were 79%, 78%, 21%, 7%, and 36% in the POmT, PcrV, OprF, mTox, and alum-alone groups, respectively. Significant improvement in acute lung injury and reduction in acute mortality within 24 h after infection was observed in the POmT and PcrV groups than in the other groups. Overall, the POmT vaccine exhibited efficacy comparable to that of the PcrV vaccine. The future goal is to prove the efficacy of the POmT vaccine against various P. aeruginosa strains.


Introduction
Pseudomonas aeruginosa, a gram-negative aerobic bacillus, rarely causes serious infections in healthy individuals but causes opportunistic infections in immunocompromised patients [1,2]. In immunocompromised patients, P. aeruginosa is the causative agent of skin infections following severe burns and ventilator-associated pneumonia in intensive care settings. Since the 1970s, research has been conducted on immunotherapy using vaccines as an effective prophylactic means to replace antibacterial drug therapy [3,4]. Multidrug-resistant P. aeruginosa has emerged in recent years [5][6][7], but active and passive immunotherapy that does not rely on antibacterial agents for P. aeruginosa infection has not yet reached clinical application.
To date, we have demonstrated the active immunization effect of V antigen (PcrV) targeting the type III secretion system involved in P. aeruginosa virulence, as well as passive immunization using a specific antibody (anti-PcrV antibody) that blocks the PcrV antigen [8]. P. aeruginosa uses its type III secretion system to inject four type III secretory toxins (ExoS, ExoT, ExoU, ExoY) into target eukaryotic cells, thereby exerting pathogenic toxicity [9]. Among these toxins, ExoU, a phospholipase toxin, causes cell damage and  Supplementary Table S1. The enzymatic active glutamic acid at #553 of toxA#4 deleted by a specific PCR oligonucleotide (toxA_D553) to generate a non-catalytic mutated A fragment (mToxA#406-#613(E553Δ)). The DNA sequences of PcrV, OprF#190-#342, and mToxA#4 were connected to glycine-serine short linker oligonucleotide sequences to create a trivalent protein antigen designated POmT (which stands for a conjugate of PcrV and part and mutated exotoxin A). The PCR amplicons were ligated to the protein expression vecto the expression and purification of recombinant proteins, the amino-terminal hexahistidin the proteins were removed using exopeptidase.

Immunizations
Certified pathogen-free male ICR mice (4 weeks old; body weight, 20-25 g) ( Laboratory Supplies, Co., Ltd., Kyoto, Japan) were housed in cages (with filter top pathogen-free conditions. All animal experiments were performed under the app the Animal Research Committee of the Kyoto Prefectural University of M (Approval numbers: M2019-563, M2020-314, M2021-335, M2022-326). One experiments consisted of 10-15 mice from three vaccination groups, and several s repeated on different days to check the reproducibility of the effects of vaccinatio  Supplementary Table S1. The enzymatic active glutamic acid at #553 of toxA #406-#613 was deleted by a specific PCR oligonucleotide (toxA_D553) to generate a non-catalytic mutated exotoxin A fragment (mToxA #406-#613(E553∆) ). The DNA sequences of PcrV, OprF #190-#342 , and mToxA #406-#613(E553∆) were connected to glycine-serine short linker oligonucleotide sequences to create a synthetic trivalent protein antigen designated POmT (which stands for a conjugate of PcrV and parts of OprF and mutated exotoxin A). The PCR amplicons were ligated to the protein expression vectors. After the expression and purification of recombinant proteins, the amino-terminal hexahistidine tags of the proteins were removed using exopeptidase.

Immunizations
Certified pathogen-free male ICR mice (4 weeks old; body weight, 20-25 g) (Shimizu Laboratory Supplies, Co., Ltd., Kyoto, Japan) were housed in cages (with filter tops) under pathogen-free conditions. All animal experiments were performed under the approval of the Animal Research Committee of the Kyoto Prefectural University of Medicine (Approval numbers: M2019-563, M2020-314, M2021-335, M2022-326). One set of experiments consisted of 10-15 mice from three vaccination groups, and several sets were repeated on different days to check the reproducibility of the effects of vaccination. The recombinant proteins (10 µg/dose), used at a previously reported effective antigen dose [8], were formulated in combination with aluminum hydroxide gel (alum, 20 mg/mL, 100 µL/dose, Alhydrogel adjuvant 2%, vac-alu-250, InvivoGen, Toulouse, France). Aluminum hydroxide gel solution was added into the solution containing one vaccine protein or a combination of multiple proteins at a final volume ratio of 1:1 and was mixed well by pipetting the solution up and down for at least 5 min. The solution was diluted to 200 µL/dose with saline. On day 0, eight groups of mice were vaccinated subcutaneously on their backs with the vaccine adjuvant conjugate. In addition, one group of mice was injected with alum alone (Table 1). Twenty-eight days later, booster immunizations or injections were administered using the same formulations. 2.3. Anti-PcrV, Anti-OprF, Anti-ToxA, and Anti-POmT Titer Measurements On 22 days after the final immunization (On day 50 from the first immunization), in the vaccinated mice, the peripheral blood was collected to determine specific IgG titers to the vaccine proteins by enzyme-linked immunosorbent assay (ELISA), as reported previously [25].

Infectious Challenge by P. aeruginosa
The P. aeruginosa PA103, originally from an Australian patient [26], is a cytotoxic strain with positive type III secretion of ExoT and ExoU. PA103∆pcrV, a mutant lacking the pcrV gene, was previously produced by the Dara W. Frank laboratory (Department of Microbiology and Immunology, Medical College of Wisconsin) [8]. Bacterial suspensions were prepared, as reported previously [25,27]. On day 56 (28 days after the final immunization, 12 weeks old), the solution containing either PA103 (1.0 × 10 6 cfu in 60 µL of saline) or PA103∆pcrV (1.0 × 10 8 cfu in 60 µL of saline) was instilled into the lung of each vaccinated mouse through an endotracheal needle, as described previously under light inhalational anesthesia using sevoflurane [27]. PA103∆pcrV was administered at a 100-fold higher dose than PA103. Mice in the saline (no infection) group were given saline intratracheally only. The survival and body temperatures of all mice were monitored for 24 h, after which the survivors were euthanized. As we reported previously, the mice which received a lethal dose of P. aeruginosa PA103 (1.0 × 10 6 cfu, LD 50%~2 4 h) became hypothermic within 4 h [8,10]. The hypothermic pathology and mortality within 24 h in this mouse model of pneumonia is not a result of bacterial multiplication in the infected organ but a consequence of cell necrosis of lung epithelial cells due to the translocation or the type III secretory toxin ExoU of P. aeruginosa into the lung epithelial cells within 4 h after infection [28]. We have also reported that the necrosis of lung epithelial cells due to ExoU toxin translocation occurs within 1 h after infection in an in-vitro culture cell model [10]. In addition, it has been reported that this pathology disappears after infection with an isogenic mutant strain lacking the enzymatically active ExoU of P. aeruginosa [10]. Therefore, our prophylactic model focuses on the first 4 to 24 h after infection. Accordingly, the experiment was completed in 24 h to focus on the pathogenesis of acute lung injury induced by P. aeruginosa, in addition to considerations on animal welfare and preventing contamination within the facility according to the infection experiment regulations of our facility. It is worth noting that the reasons for the different numbers of animals in each group are as follows. In a single experiment, a maximum of 15 mice were used, including the POmT group as a positive control (effective) group and the alum group as a negative control (ineffective) group, with other groups to be investigated and interposed in between to determine the effect. Data were obtained for more than 10 animals/group, with experiments repeated at least three times to confirm reproducibility. Therefore, in total, the PA103 infection series comprised 42 animals in the POmT group and 28 animals in the alum group, which were both larger than the other groups (10-18 animals). The lungs of each mouse were collected and homogenized using a homogenizer (Polytron PT10/35, Kinematica, Luzern, Switzerland) for further evaluation.

Lung Edema Index, Myeloperoxidase (MPO) Activities, and Bacteriological Assay
The wet-to-dry weight ratio of the lungs was measured as an index of lung edema at the 24-h time point, as described previously [28]. MPO activity in the lung homogenates was measured, as reported previously [25]. The sequentially diluted lung homogenate was inoculated on a sheep blood agar plate and incubated at 37 • C overnight to calculate the number of remaining bacteria in a gram of lung tissues.

Histopathological Assay
Lung tissue was collected at 24 h after infection from surviving mice, two mice per group, and subjected to histological evaluation. The lungs were perfused with 10% formalin neutral buffer solution for fixation and embedded in paraffin. The mounted sections were stained with hematoxylin-eosin.

Statistical Analysis
For multiple comparisons of survival curves, the post-hoc analysis for the log-rank test was used with p-values adjustment by the Benjamini & Hochberg method using RStudio (2022.7.1. Build 554, R version 4.2.1, RStudio PBC). One-way analysis of variance and Bonferroni's test (Prism 9, GraphPad Software, La Jolla, CA, USA) were used to compare IgG titers, body temperatures, the lung edema index, and the MPO assay data. The nonparametric tests (Kruskal-Wallis test) and the multiple comparison tests (Dunn's test) were used to compare the number of remaining lung bacteria with a statistical significance (p-value < 0.05).

Specific Antibody Titers Induced by Vaccination
In the first series of experiments in which vaccinated mice were eventually infected with P. aeruginosa PA103 (Figure 3a), serum anti-POmT IgG titers in mice vaccinated against POmT, PcrV, OprF, or mTox were statistically significantly increased compared with the control titers ( † p < 0.01). Anti-PcrV IgG titers from mice vaccinated against POmT or PcrV, anti-OprF IgG titers from mice vaccinated against POmT or OprF, and anti-mTox IgG titers from mice vaccinated against POmT or mTox were significantly increased ( † p < 0.01). In the second series of experiments, in which vaccinated mice were later infected with the isogenic mutant PA103∆pcrV strain (Figure 3b), vaccinated mice exhibited a similar antibody titer profile as those in the first series of experiments.

Specific Antibody Titers Induced by Vaccination
In the first series of experiments in which vaccinated mice were eventually infected with P. aeruginosa PA103 (Figure 3a), serum anti-POmT IgG titers in mice vaccinated against POmT, PcrV, OprF, or mTox were statistically significantly increased compared with the control titers ( † p < 0.01). Anti-PcrV IgG titers from mice vaccinated against POmT or PcrV, anti-OprF IgG titers from mice vaccinated against POmT or OprF, and anti-mTo IgG titers from mice vaccinated against POmT or mTox were significantly increased ( † < 0.01). In the second series of experiments, in which vaccinated mice were later infected with the isogenic mutant PA103ΔpcrV strain (Figure 3b), vaccinated mice exhibited similar antibody titer profile as those in the first series of experiments.   Mice were subcutaneously immunized twice on their back on days 0 and 28. On day 50, a small volume of peripheral blood was collected from the tail vein of each mouse, and the specific titers of vaccine antigens were measured in each serum sample. Then, on day 56, each mouse underwent tracheal instillation of P. aeruginosa PA103 (1.0 × 10 6 cfu/mouse) or its pcrV-deleted isogenic mutant PA103ΔpcrV (1.0 × 10 8 cfu/mouse), and their body temperature and survival were monitored for 24 h.

Specific Antibody Titers Induced by Vaccination
In the first series of experiments in which vaccinated mice were eventually infected with P. aeruginosa PA103 (Figure 3a), serum anti-POmT IgG titers in mice vaccinated against POmT, PcrV, OprF, or mTox were statistically significantly increased compared with the control titers ( † p < 0.01). Anti-PcrV IgG titers from mice vaccinated against POmT or PcrV, anti-OprF IgG titers from mice vaccinated against POmT or OprF, and anti-mTox IgG titers from mice vaccinated against POmT or mTox were significantly increased ( † p < 0.01). In the second series of experiments, in which vaccinated mice were later infected with the isogenic mutant PA103ΔpcrV strain (Figure 3b), vaccinated mice exhibited a similar antibody titer profile as those in the first series of experiments.

Survival Rates and Body Temperatures
All groups of mice, except the saline-treated control mice, were infected intratracheally with PA103 (1.0 × 10 6 cfu) or PA103ΔpcrV (1.0 × 10 8 cfu) on day 56 after the first immunization, and their survival and body temperature were monitored for 24 h (Figure 4).

Survival Rates and Body Temperatures
All groups of mice, except the saline-treated control mice, were infected intratracheally with PA103 (1.0 × 10 6 cfu) or PA103∆pcrV (1.0 × 10 8 cfu) on day 56 after the first immunization, and their survival and body temperature were monitored for 24 h (Figure 4).

Comparison between the POmT and Monovalent Vaccines
First, the POmT group was compared with the one-antigen vaccine (PcrV, OprF, or mTox) groups. The 24-h survival rates were 79%, 78%, 21%, 7%, and 36% in the POmT, PcrV, OprF, mTox, and alum-alone groups, respectively (Figure 4a). Statistically, the POmT and PcrV group had significantly higher survival rates than the alum-alone group ( † p < 0.001 and † p = 0.019, respectively). The survival rate in the mTox group was significantly lower than those in the alum alone ( † p = 0.012) and PcrV ( ‡ p = 0.0002) groups. The body temperature in surviving mice 12 h after PA103 administration was 34.3 ± 2.2 • C in the POmT group, 33.7 ± 2.3 • C in the PcrV group, 33.6 ± 2.7 • C in the OprF group, 29.6 ± 2.4 • C in the mTox group, and 31.1 ± 3.1 • C in the alum-alone group. Body temperatures at 12 h significantly differed between the POmT and alum-alone groups ( † p < 0.001). There was no statistically significant difference in survival or body temperature between POmT and PcrV groups.

Comparison of POmT and Bivalent Combination Vaccines
Second, the two-antigen mixed vaccine groups (PcrV+OprF, PcrV+mTox, or OprF+mTox) were evaluated (Figure 4b). The 24-h survival rate was 70% in the PcrV+OprF group, 67% in the PcrV+mTox group, 9% in the OprF+mTox group, 79% in the POmT group, and 36% in the alum-alone group. The survival rates were significantly higher in the PcrV+OprF and PcrV+mTox groups than in the OprF+mTox group ( ‡ p = 0.006 and ‡ p = 0.006, respectively). The survival rate in the OprF+mTox group was significantly lower than in the POmT group ( ‡ p < 0.0001). Thus, retention of the PcrV antigen component in the vaccine was critical for protective efficacy. At 12 h after PA103 administration, the body temperature of the surviving mice was 34.8 ± 2.0 • C in the PcrV+OprF group ( § p = 0.013 vs. OprF+mTox), 33.7 ± 2.4 • C in the PcrV+mTox group, and 28.2 ± 0.2 • C in the OprF+mTox group. Thus, the presence of the PcrV antigen from the vaccine was critical for preventing infectious hypothermia following PA103 infection.

Comparison of POmT and the Trivalent Combination Vaccine
Third, the three-antigen mixed vaccine (PcrV+OprF+mTox) group was compared with the POmT group (Figure 4c). There was no significant difference in the survival rate 24 h after PA103 administration between these groups (79% vs. 70%). At 8 h after bacterial administration, the body temperature in the surviving mice was 32.2 ± 2.3 • C in the POmT group and 32.9 ± 1.8 • C in the PcrV+OprF+mTox group. At 12 h after infection, the body temperature was 34.3 ± 2.2 • C in the POmT group and 34.3 ± 1.5 • C in the PcrV+OprF+mTox group. At 12 h, the body temperature was significantly higher in the POmT and PcrV+OprF+mTox groups than in the alum-alone group ( † p < 0.001 and † p = 0.016, respectively). However, body temperature did not differ between the POmT and PcrV+OprF+mTox groups over 24 h.

Comparison between the POmT and PcrV Monovalent Vaccine Groups following Infection with P. aeruginosa PA103∆pcrV
The PA103∆pcrV strain lacks type III secretory toxicity because of an artificial genetic defect in PcrV. Therefore, pulmonary injury and death following pulmonary infection by PA103∆pcrV are attributable to pathogenicity other than type III secretory toxicity. However, the lethal dose of the PA103∆pcrV strain in a mouse lung injury model is approximately 100-fold higher than that of the wild-type strain PA103. Therefore, we compared the effects of the POmT and PcrV vaccines on the survival of mice infected with the PA103∆pcrV strain (Figure 4d). The survival rate of PcrV-vaccinated mice infected with PA103∆pcrV was 47%, which was not significantly lower than that of uninfected mice (p = 0.23), while the survival rates in the alum and POmT groups were 50% and 68%, respectively. Although the survival rate with POmT was higher than that with PcrV, this difference was not statistically significant (p = 0.23). The survival rates of PA103 (1 × 10 6 cfu)-infected mice treated with POmT, PcrV, and alum were 79%, 78%, and 36%, respectively, while the survival rates of PA103∆pcrV (1 × 10 8 cfu)-infected mice treated with POmT, PcrV, and alum were 68%, 47%, and 50%, respectively. Although the bacterial dose of PA103∆pcrV was 100 times higher than that of PA103, there was no statistically significant difference in mortality between PA103-infected and PA103∆pcrV-infected mice and the corresponding vaccine groups.

Lung Edema, MPO Assay, Bacteriology, and Lung Histology
The severity of lung edema was measured in mice infected with the PA103 and PA103∆pcrV strains (Figure 5a). Following PA103 administration, all mice exhibited severe lung edema compared with uninfected control mice (3.3 ± 0.2, * p < 0.002). The lung edema index was 5.4 ±1.4 in the POmT group and was the highest in the OprF+mTox group (6.6 ± 0.8), although there was no statistically significant difference among the groups infected with PA103. There were also no significant differences among the POmT, PcrV, and alum-alone groups following P. aeruginosa PA103∆pcrV administration.

Lung Edema, MPO Assay, Bacteriology, and Lung Histology
The severity of lung edema was measured in mice infected with the PA PA103ΔpcrV strains (Figure 5a). Following PA103 administration, all mice e severe lung edema compared with uninfected control mice (3.3 ± 0.2, * p < 0.002). T edema index was 5.4 ±1.4 in the POmT group and was the highest in the Opr group (6.6 ± 0.8), although there was no statistically significant difference am groups infected with PA103. There were also no significant differences among the PcrV, and alum-alone groups following P. aeruginosa PA103ΔpcrV administration The MPO activities at 24 h in P. aeruginosa-infected lungs were quantified to e the severity of inflammation (Figure 5b). The alum-alone, POmT, PcrV, OprF, an groups showed significant increases in MPO activity compared with the uninfecte (* p < 0.0001-0.008). Among the PA103-infected groups, MPO activity was sign lower in the POmT and PcrV groups than in the alum-alone group ( † p < 0.0001 a 0.002, respectively).
Total bacterial counts in the lungs were calculated in both surviving and de 24 h after P. aeruginosa infection ( Figure 6). Following PA103 infection, lung b counts were significantly lower in the PcrV+OprF+mTox group than in the alu group (Figure 6a) ( † p = 0.006). No significant differences were observed between t groups and the alum-alone group. Following PA103ΔpcrV infection, the nu bacteria was lower in the POmT group than in the PcrV and alum groups, albeit statistical significance (Figure 6b). More mice in the group immunized with POm found to have a decreased number of bacteria in their lungs relative to the initial b dose (cfu/gram of lung tissue) ( § p < 0.05) (red dashed lines, Figure 6a). The MPO activities at 24 h in P. aeruginosa-infected lungs were quantified to evaluate the severity of inflammation (Figure 5b). The alum-alone, POmT, PcrV, OprF, and mTox groups showed significant increases in MPO activity compared with the uninfected group (* p < 0.0001-0.008). Among the PA103-infected groups, MPO activity was significantly lower in the POmT and PcrV groups than in the alum-alone group ( † p < 0.0001 and † p = 0.002, respectively). Total bacterial counts in the lungs were calculated in both surviving and dead mice 24 h after P. aeruginosa infection ( Figure 6). Following PA103 infection, lung bacterial counts were significantly lower in the PcrV+OprF+mTox group than in the alum-alone group (Figure 6a) ( † p = 0.006). No significant differences were observed between the other groups and the alum-alone group. Following PA103∆pcrV infection, the number of bacteria was lower in the POmT group than in the PcrV and alum groups, albeit without statistical significance (Figure 6b). More mice in the group immunized with POmT were found to have a decreased number of bacteria in their lungs relative to the initial bacterial dose (cfu/gram of lung tissue) ( § p < 0.05) (red dashed lines, Figure 6a). Histological changes were evaluated in the mouse lungs 24 h post-infection. Destruction of the alveolar structure was observed with enhanced neutrophil infiltration and alveolar hemorrhage in the lungs of mice in the alum-alone, OprF, and mTox groups (Figure 7). On the other hand, the lungs of POmT-vaccinated mice and PcrV-vaccinated mice displayed significantly fewer inflammatory changes than those of the other groups. It is assumed that lung histology showed less inflammation than the average for the group because lung tissue was collected from surviving mice over 24 h in each group. These histological findings roughly correlated with the MPO assay results.  Histological changes were evaluated in the mouse lungs 24 h post-infection. Destruction of the alveolar structure was observed with enhanced neutrophil infiltration and alveolar hemorrhage in the lungs of mice in the alum-alone, OprF, and mTox groups (Figure 7). On the other hand, the lungs of POmT-vaccinated mice and PcrV-vaccinated mice displayed significantly fewer inflammatory changes than those of the other groups. It is assumed that lung histology showed less inflammation than the average for the group because lung tissue was collected from surviving mice over 24 h in each group. These histological findings roughly correlated with the MPO assay results. Histological changes were evaluated in the mouse lungs 24 h post-infection. Destruction of the alveolar structure was observed with enhanced neutrophil infiltration and alveolar hemorrhage in the lungs of mice in the alum-alone, OprF, and mTox groups (Figure 7). On the other hand, the lungs of POmT-vaccinated mice and PcrV-vaccinated mice displayed significantly fewer inflammatory changes than those of the other groups. It is assumed that lung histology showed less inflammation than the average for the group because lung tissue was collected from surviving mice over 24 h in each group. These histological findings roughly correlated with the MPO assay results.

Discussion
Over nearly half a century, several P. aeruginosa virulence factors that are potential vaccine antigens have been identified and investigated as candidate vaccine formulations. However, all vaccine trials using these virulence factors have ended without satisfactory results that could lead to clinical use [4]. OprF/I, an outer membrane protein, is highly immunogenic and expressed by all P. aeruginosa serotypes. A vaccine containing this outer membrane protein (IC43) exhibited a strong safety profile in a preclinical study, eliciting antibodies that promote complement-dependent opsonization of P. aeruginosa, leading to a high degree of efficacy against P. aeruginosa infection [29]. Based on its protective effect, it was advanced to Phase III trials [30,31]. Although good immunogenicity and safety were observed, the mortality and infection rates showed no difference from those in the placebo group. Based on its potent cytotoxicity, exotoxin A was identified as a cytotoxic factor in the pathogenicity of P. aeruginosa and was proposed as a candidate vaccine antigen. However, few studies have tested exotoxin A alone as a vaccine antigen. Both passive and active immunization were previously used for antigens in which exotoxin A is conjugated to LPS, and neither approach was successful [32][33][34]. However, vaccines against exotoxin A have not been applied clinically to date.
The type III secretion system of gram-negative bacteria accomplishes the direct delivery of protein toxins from bacterium to host by nanosyringe "injectisomes," which form a conduit across the two bacterial membranes, extracellular space, and the plasma membrane of a target eukaryotic cell [9]. PcrV, which has a cap-like structure at the tip of the needle-like structure of the P. aeruginosa injectisome, is involved in the toxin translocation mechanism across the plasma membrane of a target eukaryotic cell [11][12][13]. In our previous research, we found that a specific antibody against PcrV suppressed the toxicity of the type III secretion system [11]. A clinical trial of this antibody has been conducted, but it has not yet been put to practical use [35][36][37]. Because P. aeruginosa carries a variety of virulence factors, studies have been conducted on vaccines focusing on each virulence factor, but none have been applied clinically. Recently, the effectiveness of vaccines (active immunization) with bivalent or trivalent antigens has been investigated [38][39][40][41] to broaden the spectrum of prophylactic efficacy against P. aeruginosa subspecies by targeting multiple virulence factors rather than a single virulence factor. To develop a multivalent P. aeruginosa vaccine, inhibition of the type III secretion system, the major virulence factor of P. aeruginosa, is of utmost importance. Based on these reports, we focused on the pathogenic diversity of P. aeruginosa, including PcrV. We decided to create a multivalent vaccine, which included the following components: (1) The type III secretion system PcrV of P. aeruginosa exerts pathogenicity by injecting toxin proteins into target cells using the secretion apparatus; (2) OprF is an outer membrane protein expressed in all P. aeruginosa serotypes and is effective in both active and passive immunization; (3) The type III toxin secretion system causes cell death by inhibiting protein synthesis in target cells, and 70%-90% of clinical strains are reported to be exotoxin A-producing strains [18,19]. We compared the active immunization efficacy of a novel vaccine POmT conjugated with three monovalent antigens using a monovalent vaccine containing the conventional monovalent vaccines PcrV, OprF, and mTox and a mouse model of P. aeruginosa pneumonia.
In this study, significant differences were observed between the POmT vaccine group and the alum-alone group (control group) regarding the 24-h survival rate after PA103 strain infection and the change in body temperature 12 h after infection. In contrast, there was no significant difference in any variable between the POmT vaccine and any vaccine containing PcrV. These findings indicate that the binding of OprF and exotoxin A to PcrV does not eliminate the vaccine effect of PcrV, that is, the inhibitory effect of the type III secretion system, and the POmT vaccine antigen is active against acute lung injury caused by P. aeruginosa. In addition, in comparison to serum IgG, it was revealed that antibody titers against OprF and exotoxin A increased steadily following immunization with the novel tri-antigen POmT vaccine to the same level as achieved with immunization by individual protein antigens. However, immunization with OprF alone, mTox alone, and OprF+mTox resulted in a lower survival rate than that in the alum group. It remained unknown whether immunization with OprF or mTox brought about an adverse immune effect on survival or whether the lower survival rate was due to the instability of the experiment. However, the OprF, mTox alone, and bivalent OprF+mTox vaccines did not demonstrate the positive immune effects of PcrV and POmT.
The aforementioned results using P. aeruginosa strain PA103 confirmed that the type III secretion system is significantly involved in acute lung injury caused by P. aeruginosa. To evaluate the protective effect of the POmT vaccine against pathogens other than the type III secretion system, we conducted an infection experiment using the pcrV deletion mutant PA103∆pcrV, which lacks a functional type III secretion system and compared it with the POmT vaccine [8]. No significant difference was observed between the POmT and PcrV vaccine groups. These findings indicate that the pathogenic mechanism of acute lung injury induced by P. aeruginosa is highly dependent on the type III secretion system and is largely unrelated to other pathogenic factors. Previous studies on vaccine efficacy against P. aeruginosa LPS and OprF were planned before the discovery of the P. aeruginosa type III secretion system and ExoU toxin, an effector molecule representing its toxicity, in the mid-1900s. As our results suggest, the ability to counteract P. aeruginosa type III secretion system-induced toxicity is essential for preventing and treating acute pulmonary P. aeruginosa infections. However, it remains possible that vaccines can be effective against P. aeruginosa infectious pathologies dependent on other virulence factors. At least in the present study, the POmT vaccine showed significantly higher survival rates in a lethal P. aeruginosa pneumonia model, suggesting that the POmT vaccine has the potential to be effective in preventing a more comprehensive range of P. aeruginosa infections. Finally, in this experiment, all prepared component vaccines were administered subcutaneously to evaluate the protective effect of the POmT vaccine with those administered using more conventional immunization methods. P. aeruginosa is a pathogen that infects the respiratory tract, so airway mucosal immunity is essential. As we recently reported the efficacy of intranasal administration of the PcrV-CpG deoxyoligonucleotide vaccine [25], our future studies will focus on confirming the effectiveness of intranasal administration of the POmT vaccine.
Since our discovery in 1999 of the immune effect of the PcrV antigen against P. aeruginosa pneumonia [8], we have been working on developing PcrV-targeted therapeutic antibodies and PcrV vaccines. However, since P. aeruginosa retains diverse pathogenic mechanisms and causes various infectious diseases [5], many pharmaceutical companies have discouraged the development of immunotherapies targeting only a single PcrV antigen, mainly because of its potential narrow spectrum against various P. aeruginosa pathogenicities. No vaccine against P. aeruginosa has been clinically applied to date [4]. Since there is no evidence that a multi-antigen vaccine is superior to a PcrV single-antigen vaccine in P. aeruginosa infection, we investigated this by comparing vaccines targeting only PcrV with vaccines that include other antigens. Unfortunately, as described above, POmT, a trivalent vaccine, failed to show a definite advantage over the mono-antigen PcrV vaccine. However, the findings of this experiment revealed several important points. First, we confirmed that P. aeruginosa type III secretory toxicity and its inhibition are critical factors in the etiology and prevention of P. aeruginosa-induced acute lung injury, with other virulence factors playing a minor role. Second, the three antigens, artificially linked via an amino-acid linker in the molecular structure, retained the antigenicity and immune effects of PcrV. This finding was significant because, in producing vaccines containing three separate components, manufacturing regulations require that safety and efficacy tests are conducted for each element, including antigens and adjuvants [42], complicating the development of multi-antigen vaccines and raising costs considerably. Therefore, combining antigens at the gene level to generate a single molecule is considered the most realistic option when manufacturing a multiprotein antigen vaccine. As mentioned above, there is a concern that vaccines that do not contain PcrV (OprF and/or mTox), at least in our P. aeruginosa pneumonia model, may exacerbate rather than improve lung injury. In the case of immunotherapies against virus infections, the possibility that immunization against specific antigens that do not suppress toxicity exacerbates cellular damage, especially of phagocytic cells, has been reported as the antibody-dependent enhancement (ADE) phenomenon [43]. This mechanism of ADE involves the interaction of pathogen-antibody immune complexes to phagocytic cells through the adherence of the antibody Fc region with cellular Fc receptors. In this phenomenon, non-neutralizing antibodies generated by vaccination and bound to pathogen proteins can promote the subsequent contact of pathogens to the target cells and intensify the inflammatory process during infection. Therefore, antibodies derived from vaccines that do not suppress cytotoxicity may promote the interaction of pathogens with phagocytic cells and eventually induce more severe cytotoxicity. A mechanism similar to the ADE phenomenon may help to explain the poor performance of the present PcrV-free OprF and/or mTox vaccine.

Conclusions
This study created POmT as a single recombinant protein by combining PcrV, OprF, and mTox with glycine-serine linkers. In terms of preventing acute lung injury and subsequent death due to P. aeruginosa infection, immunization with POmT demonstrated comparable prophylactic levels with PcrV immunization according to all tested parameters, such as specific antibody titers, survival, lung edema, MPO, bacteriology in the lungs, and lung histology. In addition, the protective effects of the POmT vaccine were almost the same as those observed with the mixture of PcrV, OprF, and mTox. In our mouse model series, we could not demonstrate the benefit of including OprF and/or mTox as vaccine components against P. aeruginosa pneumonia. We confirmed that immunity to PcrV is essential for preventing P. aeruginosa-induced acute lung injury and that immunity to OprF and exotoxin A has little effect. In addition, when using genetic recombination technology, even with the artificially synthesized trivalent vaccine, antibody titers increased. Moreover, the impact on PcrV, without affecting the antigenicity of the components, including PcrV, was confirmed. Whether the P. aeruginosa trivalent vaccine POmT is effective against other P. aeruginosa strains and different P. aeruginosa infections is an issue that will be addressed in future studies.

Data Availability Statement:
The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request.